The MaxDiscovery™ ß-Galactosidase ELISA Kit is an enzyme immunoassay that analyzes the quantity of ß-galactosidase in cells, tissues or serum. ß-galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of ß-galactosides into monosaccharides. The ß-galactosidase protein is a large (120 kDa, >1000 amino acids) protein that forms a tetramer. In E. coli the ß-galactosidase protein is encoded by the lacZ gene which is part of the lac operon. The enzyme's function in E. coli is to cleave the disaccharide lactose to form glucose and galactose so they can be used as carbon/energy sources. The lacZ gene from E. coli is one of the most commonly used reporter genes for testing the efficiency of expression vector mediated gene transfer and for studying the regulation of gene promoters. ß-galactosidase is not typically expressed in mammalian cells, however; ß-galactosidase positive staining is a marker for mammalian cellular senescence. ß-galactosidase is a useful reporter gene both in cell culture and in vivo applications due to its ease of detection. Like most ELISA assays, the MaxDiscovery™ ß-Galactosidase ELISA test relies on a Horseradish peroxidase (HRP) conjugated primary antibody and the TMB (3,3´,5,5´-tetramethylbenzidine) substrate. TMB is a chromogen that yields a blue color when oxidized with hydrogen peroxide (catalyzed by HRP) and has a major absorbance at 370 nm and 652 nm. The color then changes to yellow with the addition of acid with maximum absorbance at 450 nm. The relative amount of ß-galactosidase protein in the cells will be directly proportional to the amount of signal that is obtained and thus is an excellent technique for detecting protein expression within cell lines.
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